1. Field of the Invention
The present invention relates to embryonic stem cell-derived cardiomyocytes, and a cellular therapeutic agent comprising the same as an active ingredient. More particularly, the present invention relates to a method for producing embryonic stem cell-derived cardiomyocytes, cardiomyocytes produced by the method, a method for producing cardiomyocyte bodies from the cardiomyocytes, cardiomyocyte bodies produced by the method, a cellular therapeutic agent comprising the cardiomyocyte bodies as an active ingredient for the treatment of cardiac diseases, and a method for treating cardiac diseases using the cellular therapeutic agent.
2. Description of the Related Art
Embryonic stem cell (ESC)-derived cardiomyocytes (CM) can be used in regenerative therapies for cardiovascular diseases, and thus a variety of techniques for differentiation and isolation of CMs have been developed by many researchers. However, these methods face difficulties in their practical applications due to a low yield of CMs. CMs have been isolated from contracting human embryoid bodies (hEBs) by microdissection. It has been known that formation of hEBs induces generation of contracting CMs from undifferentiated hESCs, but there are a few drawbacks to CM generation by this method. First, hEBs do not show a unique splitting pattern of three germ layers in three-dimensional culture. Second, the differentiated cells are mixed with cells of different cell lineages. Third, it is difficult to isolate CMs in a high concentration. Therefore, many researchers have tried to isolate a large amount of CMs by mechanical isolation, Percoll density gradient isolation, and by use of KDR, CD15 and CD16.
However, pure CM cells cannot be completely isolated by these methods, and thus there is still a drawback in their clinical application. It is also difficult to apply the conventional culture method in CM isolation due to a lack of CM-specific surface markers. In order to overcome these problems, MLC-2v-induced-GFP-expressing hESCs were produced by transduction using a recombinant lentiviral vector system, and hESCs were used for the isolation of CMs from hEBs by FACS isolation. However, this clinical application of hESCs is highly restricted because viral DNA can be integrated into DNA in the body. In addition, isolation of CMs from contracting hEBs by microdissection has a problem that CMs are mixed with other endodermal lineage cells because of their characteristic of coexisting with endodermal cells rather than ectodermal cells, and many studies have been actively conducted to solve this problem.
On the other hand, it is also an important factor to obtain a sufficient amount of CMs to be used as therapeutic agents for cardiovascular diseases, because mature CMs have limited proliferative activity. For this work, various differentiation methods using growth factors or an END-2 co-culture system with an endodermal cell line providing CM proliferation and differentiation have been developed. However, this method has financially costly due to the use of growth factors, and the inconvenience of further isolating CMs from END-2 cells co-cultured therewith. Accordingly, there is a demand for production and isolation methods of CMs which are able to provide high efficiency and yield for cell-based therapies for cardiac diseases.
Under this background, the present inventors have made many efforts to develop a method for purifying ESC-differentiated cardiomyocytes with high yield and efficiency. As a result, they developed a method for purifying ESC-differentiated cardiomyocytes using a serum-free medium, and found that cardiomyocyte bodies (CBs) to be used for the treatment of cardiac diseases can be produced by suspension-culture of the purified CMs in the serum-free medium, thereby completing the present invention.